Efforts to exploit the dependence of tumors of fatty acids (FA) have thus far focused only on inhibiting FA synthesis. We have demonstrated that, in addition to the lipogenic apparatus, tumors are equipped to acquire circulating, diet-derived FAs using a lipolytic pathway. This employs the secreted enzyme lipoprotein lipase (LPL), which releases FAs from lipoproteins, and CD36, the channel for FA uptake. Alternatively, LPL may act as a nonenzymatic linker to facilitate lipoprotein endocytosis by binding to a cell surface heparin sulfate proteoglycan. We have demonstrated brisk LPL expression in all tumors examined to date (n=181). Our overarching hypothesis is that LPL-mediated FA uptake provides alternative mechanisms for FA acquisition by tumors that fuel growth and metastasis. We propose four Aims to test this idea in vivo. First we will define the role of LPL in the PyMT mouse breast cancer model combined with LPL-KO. PyMT tumors express abundant LPL and metastasize to lung, a tissue rich in LPL. Mice on low- or high-fat diets will be assessed for tumor growth and metastasis, and the phospholipid composition of tumor plasma membranes will be comprehensively analyzed. The diets will test the idea that LPL is a key determinant of the tumor-promoting effect of dietary fat. Second, we will use xenografts of tumor cells with low and high LPL expression to examine the relevance of tumor-associated LPL, in the absence of genotype-related differences in the tumor host. The cells will also be used to determine the relative contributions of lipid synthesis vs. uptake to tumor cell anabolism using 13C-labeled substrates. Third, we will use PyMT x CD36-KO mice to examine the impact of CD36 on tumor growth and metastasis in the context of the diets. Fourth, we will determine whether the lipolytic pathway can subvert the anti-cancer effects of FA synthesis inhibition. The existence of two pathways for lipid acquisition by tumors raises the concern that targeting either one alone will be ineffective. We will administer the FA synthesis inhibitor Triclosan to PyMT mice on low- or high-fat feeding to assess this. We will also assess the impact of simultaneously targeting FA production and uptake. These studies will provide mechanistic insights into the relative importance of lipid synthesis and uptake by tumors and the impact of dietary fat on tumor biology. Overall, this work will address major questions posed by our recent discovery of a lipolytic tumor phenotype, and will provide guidance for drug development efforts aimed at clinical exploitation of the dependence of tumors on FA.